Effect of volatile compounds produced by endophytic bacteria on virulence traits of grapevine crown gall pathogen, Agrobacterium tumefaciens

The volatile organic compounds (VOCs) produced by endophytic bacteria have a significant role in the control of phytopathogens. In this research, the VOCs produced by endophytic bacteria including Serratia sp. Ba10, Pantoea sp. Sa14, Enterobacter sp. Ou80, Pseudomonas sp. Ou22, Pseudomonas sp. Sn48 and Pseudomonas sp. Ba35, which were previously isolated from healthy domesticated and wild-growing grapevine were evaluated in terms of their effects on the virulence traits of Agrobacterium tumefaciens Gh1, the causal agent of crown gall disease. Based on the gas chromatography–mass spectrometry analysis, 16, 15, 14, 7, 16, and 15 VOCs have been identified with high quality in strains of Ba10, Sa14, Ou80, Ou22, Sn48, and Ba35, respectively. All endophytic bacteria produced VOCs that significantly reduced crown gall symptoms and inhibited the populations of A. tumefaciens Gh1 at different levels. Moreover, scanning electron microscopy analysis revealed various morphological abnormalities in the A. tumefaciens cells exposed to the VOCs produced by Ba35, Ou80, and Sn48 strains. The VOCs significantly reduced swarming-, swimming-, twitching motility and biofilm formation by A. tumefaciens Gh1. Our results revealed that VOCs could reduce the attachment of A. tumefaciens Gh1 cells to root tissues of grapevine cultivars Rashe and Bidane sefid, as well as chemotaxis motility towards root extract of both cultivars. Based on our results, it was shown that the antibacterial VOCs produced by endophytic bacteria investigated in the current study can manage crown gall disease and increase our knowledge on the role of VOCs in microbial interactions.

. Analysis of variance (ANOVA) of biofilm production, swarming-, swimming-, twitching-motility, population, colony diameter and gall formation of Agrobacterium tumefaciens Gh1 under the effect of VOCs produced by endophytic bacteria. *Significant at 1% probability level. df degrees of freedom, Cv coefficient of variation.

Effect of VOCs on cell morphology. SEM analysis revealed various morphological abnormalities in the
A. tumefaciens cells exposed to the VOCs produced by Pseudomonas sp. Ba35, Enterobacter sp. Ou80, and Pseudomonas sp. Sn48 strains compared to the non-treated control (Fig. 3). The cells of the non-treated control showed normal growth, whereas more than 30% of the A. tumefaciens cells showed rough, wrinkled surfaces and cracks following co-cultivation with endophytic bacteria.

Effect of VOCs on the motility behaviors of A. tumefaciens.
According to statistical analysis in this study, significant differences were existed between the treatments in the swarming (F = 10.16; P = 0.0002), swimming (F = 7.12; P = 0.0012), and twitching (F = 15.45; P < 0.0001) assessments (Table 1). Our finding revealed that swarming motility of A. tumefaciens Gh1 significantly inhibited after exposure to VOCs of endophytic bacterial strains for 72 h. As illustrated in Figs. 4a &b, the strains Enterobacter sp. Ou80 and Pantoea sp. Sa14, with a mean of 7.5 mm showed the highest inhibition effects of A. tumefaciens Gh1 cells followed by Pseudomonas sp. Ba35, Pseudomonas sp. Ou22 and Serratia sp. Ba10 with the means of 9.1, 10.1, and 10.4 mm, respectively, as compared to the control with a mean of 14.6 mm. The VOCs produced by Enterobacter sp. Ou80, Pantoea sp. Sa14 and Serratia sp. Ba10 inhibited swimming motility of A. tumefaciens Gh1 to 8.0, 9.5, and 10.1 mm, respectively as compared to control with 15.5 mm. Correspondingly, these had the highest negative effect after 48 h ( Fig. 5a and b). Similarly, twitching motility significantly reduced to 6.4 mm by Pantoea sp. Sa14 and 6.9 mm by Enterobacter sp. Ou80 following 8.7, 9.3, and 9.9 mm by Pseudomonas sp. Ba35, Pseudomonas sp. Ou22, and Serratia sp. Ba10, respectively compared to control with 14.2 mm (Fig. 6a). Microscopic examination of the twitching motility exhibited that the circumferential colony edge of A. tumefaciens Gh1 was significantly wider in the non-exposed control compared to those exposed with the Serratia sp. Ba10, Enterobacter sp. Ou80, Pantoea sp. Sa14, and Pseudomonas sp. Ou22 volatiles (Fig. 6b).

Figure 2.
Comparison of fresh gall weight (a) and representative tumor (b) were shown on grapevine plantlets inoculated with Agrobacterium tumefaciens Gh1 (Ctrl) and A. tumefaciens cells exposed to VOCs produced by endophytic bacteria. Three replicates were used for each treatment. Error bars indicate SE of the three replicate. Different letters indicate significant differences (P = 0.05).         Table 2. Analysis of variance (ANOVA) of chemotaxis and attachment of Agrobacterium tumefaciens Gh1 towards the root exudate and the root tissue of grapevine cultivars Rashe and Bidane sefid under the effect of VOCs produced by endophytic bacteria. df degrees of freedom, Cv coefficient of variation. **Significant at 1% probability level.  (Table 3).

Discussion
Biological control of plant pathogens using beneficial bacteria can be considered as a safe and efficient method for reducing disease incidence. Biocontrol agents act through various mechanisms, and VOCs have gained increasing interest due to participating in the cross-talk between microbes and other organisms in the environment 23 . Numerous reports of VOCs produced by bacteria have previously shown the inhibition effects of bacterial plant pathogens. The VOC dimethyl disulfide produced by two rhizospheric bacteria, including Pseudomonas fluorescens and Serratia plymuthica, with antibacterial effects on two plant bacterial pathogens Agrobacterium    19,24 . In another study, the VOCs produced by Bacillus subtilis FA26, which could adversely affect the ultra-structure of cells of Clavibacter michiganenesis ssp. sepedonicus, as the causal agent of bacterial ring rot of potato have been reported 16 . In addition, some recent studies demonstrated that volatiles emitted from Bacillus strain D13 could reduce the cell motility of Xanthomonas oryzae pv. oryzae 17 . Previous report indicated that sesquiterpene albaflavenone, as a VOC compound produced by Streptomyces albidoflavus, has an antibacterial activity against Bacillus subtilis 25 . Endophytic bacteria spend their life within the plant tissues without leading to development of any disease, and they produce a wide range of volatile organic compounds with an antimicrobial activity 26 . In previous studies, endophytic bacteria were isolated from healthy domesticated and wild-growing grapevine in Iran, and some strains were detected with antagonistic effects on Agrobacterium tumefaciens, which is the causal agent of crown gall disease under both in vitro and in vivo conditions 21 . In the present study, these strains were screened for their antagonistic activity against A. tumefaciens via the production of volatile organic compounds. A. tumefaciens cells require chemotaxis, motility, and the attachment to virulence 4 . Our finding showed that VOCs produced by endophytic bacteria could significantly inhibit the growth of A. tumefaciens and crown gall symptoms. Our results reveal various inhibition effects on the chemotaxis, motility, biofilm formation, and root attachment of A. tumefaciens following the exposure to VOCs of endophytic bacteria.
Previous studies have also revealed that swimming is the most common motility behavior of A. tumefaciens and there is no evidence related to both swarming and twitching motility 27 . In contrast, our results demonstrate    28,29 . The results presented in the current study reveal that VOCs produced by endophytic bacterial strains could significantly inhibit all three forms of motility and chemotaxis. This phenomenon was further confirmed in the gall formation in planta and root attachment assay. As well, this finding is in agreement with previous studies showing that active motility and chemotaxis are required for A. tumefaciens attachment 4,27,30 . The VOCs produced by endophytic bacteria reduced the populations of A. tumefaciens Gh1. Accordingly, these results show that VOCs might keep A. tumefaciens cells away from the plants not only by inhibiting its movement and the subsequent attachment, but also by reducing its populations. It was shown that A. tumefaciens can form biofilm on abiotic and plant surfaces, as well as participating in plant tissues attachment 31 . Nonmotile mutants were significantly deficient in biofilm formation under static conditions. Under flowing conditions, however, the aflagellate mutant rapidly formed aberrantly dense, tall biofilms 27 .

RT (min) RPA (%) RT (min) RPA (%) RT (min) RPA (%) RT (min) RPA (%) RT (min) RPA (%) RT (min) RPA (%)
Our results indicate no direct relationship exists between reduction of motility and biofilm formation and the root attachment of A. tumefaciens cells exposed to VOCs emitted by individual endophytic bacterial strains. In the present research, the attachment of A. tumefaciens cells to grapevine Rashe, and Bidane Sefid cultivars was tested both in wounded and unwounded roots. Correspondingly, the obtained results indicate that A. tumefaciens attached to the grape root of both cultivars at a high population. Moreover, no significant differences were observed between the attachment to wounded and unwounded grapevine roots. This result is in agreement with the previously reported equal attachment of A. tumefaciens bv.1 to both wounded and unwounded grape roots 30 .
Electron microscopic analysis of the non-treated A. tumefaciens cells indicated normal growth, while the cells were damaged in the presence of VOCs of Enterobacter sp. Ou80, Pseudomonas sp. Ba35, and Pseudomonas sp. Sn48. Accordingly, this result is consistent with previous studies in which the abnormality of the pathogenic cells was observed after the exposure to bacterial VOCs 16,17,20 . Our previous study revealed that defense-related genes such as PR1, PR2, and PR4, were upregulated in plants treated with the strain Pseudomonas sp. Sn48 22 . This result suggests that VOCs produced by this strain could not only inhibit growth and motility traits of A. tumefaciens, but it could also induce a systemic resistance.
The GC-MS analysis showed some differences in VOCs profiles among endophytic bacterial strains. The VOCs dodecane, tetradecane, hexadecane, and eicosane were produced by all the strains tested. There have been several reports on the antibacterial and antifungal activities of these compounds [32][33][34] . The exposure to these VOCs decrease the bacterial ability to form biofilm and also bring negative effects on motility 35 . Under our experimental condition, the main VOC produced by Serratia sp. Ba10, Pantoea sp. Sa14, and Enterobacter sp. Ou80 strains was 9-Octadecenoic acid, methyl ester. Correspondingly, this fatty acid has been reported with both biosurfactant and anti-biofilm activity, so it could inhibit bacterial motility 36 . Notably, biosurfactants can reduce surface tension properties such as biofilm formation and attachment. It is suggested that the negative effects of these strains on motility and attachment of A. tumefaciens cells at least in part, are related to the production of 9-Octadecenoic acid and other fatty acids, including hexadecanoic acid methyl ester. Furthermore, these compounds have been widely described with antibacterial and antifungal activities in various studies 37,38 .
Pseudomonas sp. Ou22, Pseudomonas sp. Ba35, and Pseudomonas sp. Sn48 strains belonging to the Pseudomonas genus, produce various VOCs. Of which, the most abundant volatiles detected were long-chain alkenes such as dodecane, tetradecane, hexadecane, and aromatic hydrocarbon o-xylene, and Benzene, 1,3-dimethyl. In addition, Pseudomonas species, which are frequently reported as endophytic bacteria are well-known as plant growth-promoting bacteria by causing inhibition effects on plant pathogens 39 . Previous studies reported dodecane, tetradecane, and other VOCs released by Pseudomonas spp. with growth-promoting effect in Vigna radiate seedlings 40 . They find new insight on plant beneficial effects of VOCs produced by Pseudomonas spp. Our results reveal that VOCs of Pseudomonas sp. Ba35 strain could lead to some morphological abnormalities in A. tumefaciens cells. As well, GC-MS analysis indicated that Pseudomonas sp. Ba35 Specifically produce linalool and alpha-terpineol. Previous studies have shown that both of these compounds had strong antibacterial activity and induced the morphological change of bacteria 41-43 . In conclusion, in the present study, it was shown that VOCs produced by endophytic bacteria could inhibit motility and virulence traits of A. tumefaciens, consequently causing some morphological abnormalities in A. tumefaciens cells, as well as reducing the attachment of cells to the roots of grape. The rhizosphere is a relatively closed environment favorable for a high volatile activity. The VOCs can spread to a long-distance and then produce an antibacterial environment. Therefore, such antibacterial volatile compounds may inhibit A. tumefaciens movement in the rhizosphere, also bring negative effects on attachment, and infection of bacterial cells via root tissues. Therefore, having information on the mechanisms of antibacterial activity of these compounds is necessary to understand the microbial interactions in natural environments. MK114598), and Pseudomonas sp. Sn48 (MK114596) isolated from the domesticated and wild-growing grapevine, as well as Agrobacterium tumefaciens Gh1 (GenBank Acc. No MZ647525), which exhibited virulence in grapevine were used in this study 21 . Accordingly, these bacteria were grown on nutrient agar (NA) medium and then stored at 4-6 °C as a working stock or grown in nutrient broth (NB) medium for 24 h at 26-28 °C with shaking. Finally, sterile glycerol was added to the final concentration of 20% and then stored at -20 °C for longterm storage.

Methods
Grapevine plantlets, cultivars Rashe, and Bidane sefid were kindly provided by the department of Horticultural science, University of Kurdistan, Iran. For the collection of plantlets, all relevant permissions have been Evaluation of the antibacterial activity of VOCs produced by endophytic bacteria. The antibacterial activity of VOCs produced by endophytic bacteria against A. tumefaciens Gh1 was assessed on nutrient agar medium using a dual-culture technique. The overnight growth of the endophytic bacteria (which was adjusted to the concentration of OD 600 ≃ 1.0) was streaked on one side of the plate, while the opposite side of the plate was spot inoculated with 10 µl of the pathogen (OD 600 ≃ 0.8). In the control, the pathogen was cultured alone. Thereafter, the plates were sealed with parafilm and then incubated at 26-28 °C for 7 days. The diameter of the A. tumefaciens Gh1 colonies was measured and the colony numbers per plate were calculated as well 20 . Three replications were performed for each treatment.
Effect of VOCs on crown gall disease development. Grapevine plantlets were potted in pots containing steam-sterilized soil (consisting of 50% sand, 20% clay, 30% peat, pH 7.2). The suspension of A. tumefaciens cells with or without exposure to the VOCs of endophytic bacteria for three days at 26-28 °C was prepared in sterile water (density of OD 600 ≃ 1.0). The stems were punctuated with a sterile toothpick and 20 µl was inoculated (between the third and fourth internodes) using a sterile syringe. Plantlets were incubated in a greenhouse (95% humidity, 25-26 °C, 16 h/8 h day/night photoperiod) and gall formation was recorded up to 30 days and the fresh gall weight was measured. Notably, each treatment was tested on three separate grapevine plantlets. Swarming, swimming, and twitching motility behaviors. The motility behaviors of the A. tumefaciens Gh1 cells exposed to VOCs produced by endophytic bacteria were tested using divided Petri plates. The overnight growth of the A. tumefaciens Gh1 was adjusted to an approximate concentration of OD 600 ≃ 0.8, and then 2 µl was spotted onto one compartment of the divided plates containing NB medium plus agar (0.2-, 0.7-, 1.6%) for swimming, swarming, and twitching motility, respectively. In the other compartment, 30 µl of the endophytic bacteria with the approximate concentration of OD 600 ≃ 1.0was streaked on NA medium. The plates were incubated at 26-28 °C and the halo diameters of swarming, swimming, and twitching motility were examined after 48 and 72 h. The experiment was done in three replications 20 .

Scanning electron microscopy (SEM
Biofilm formation assay. The biofilm formation ability of A. tumefaciens Gh1 cells exposed to the VOCs produced by endophytic bacteria was investigated in polypropylene tubes. For this purpose, 10 µl of a 24-h culture of endophytic bacterial strains (OD 600 ≃ 1.0) were cultured onto one compartment of the divided plates containing NA culture medium. In the other compartment, a microtube containing 190 μl of LB liquid culture medium that was inoculated with 10 μl of A. tumefaciens Gh1 (OD 600 ≃ 0.8) was placed vertically. The plates were then sealed with parafilm and kept at 26-28 °C for 24 h. Thereafter, 25 μl of 1% crystal violet solution was added to each microtube and then kept at room temperature for 15 min. The microtubes were then rinsed twice with sterile water. Subsequently, 2 × 200 μl of 95% ethanol was added to each tube, the resulting volume was brought to 1 ml with sterile-distilled water and the absorbance was measured at 540 nm using a spectrophotometer (SPECORD 210, Analytik Jena, Germany). A. tumefaciens Gh1 cells without any exposure to VOCs were used as a control. Accordingly, the experiment was performed in a completely randomized design with three replications 44 .
Chemotaxis assay. For the chemotaxis assay, endophytic bacteria were streaked onto one compartment of the divided plates containing NA medium as described earlier. In the other plate compartment, chemotaxis buffer medium (0.1 mM EDTA, 10 mM K 2 HPO 4 , 0.35% agar, pH 7.2) was prepared, and 5 mm of the medium was also removed and then refilled with 50 µl of root extract of grapevine (cultivars Rashe, and Bidane sefid). Next, A. tumefaciens Gh1 cells were spot inoculated at a distance of 15 mm from the hole. The plates were sealed with parafilm and then incubated at 26-28 °C. The movement of the A. tumefaciens Gh1 cells towards the root extract was measured by colony diameters as well as counting the CFU/ml of the cell on NA. This experiment was performed in three replicates 45 .
Grapevine root attachment assay. www.nature.com/scientificreports/ removed and the roots were placed in 500 μl of 10 mM HEPES with pH = 7 (N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid). The obtained suspension was cultured onto NA medium and the CFU/ml was counted. Notably, three replications were considered for each treatment 30 .

Identification of VOCs produced by endophytic bacteria using GC-MS analysis.
In order to collect the VOCs produced by each endophytic bacteria, a three-compartment plate was used. One compartment, containing NA medium was streaked with 100 µl (OD 600 ≃ 1.0) overnight growth of each endophytic bacteria, the second compartment containing NA medium was spot inoculated with 5 µl (OD 600 ≃ 0.8) of A. tumefaciens Gh1, and the third compartment was filled with 0.3 g of sterile activated charcoal to adsorb the VOCs. As well, the same experimental design without endophytic bacteria or activated charcoal was used as a control. Subsequently, the plates were sealed with parafilm and then incubated at 25-26 °C for 72 h. The activated charcoal traps were transferred into glass vials and ethyl acetate (1: 1.25 W/V) was added to them. The adsorbed VOCs were extracted by shaking for 20 min, followed by the centrifugation (2500 g, 5 min) and the supernatants were analyzed by using gas chromatography device connected to a mass spectrometer (Agilent 7890B GC System / 5977A MSD). Thereafter, one microliter of the sample was injected into HP-5 ms column (30 m × 0.25 mm, 0.25 Micron), the initial column temperature was set at 60 °C, which was then increased to 260 °C at a rate of 7 °C/min, and held for 5 min. The mass spectrometer was operated in the electron ionization mode at 70 eV, with continuous scanning from 50 to 550 m/z. As well, Helium carrier gas with a purity of 99.999%, a 34 psi pressure, and a flow rate of 1 ml/min was used at this stage. The compounds were identified by comparing their mass spectra with the databases of the device, including the National Institutes of Standards and Technology (NIST) and Wiley databases, along with comparing the inhibition indices and the failure pattern reported for them 46 .
Statistical analysis. To evaluate the significance of the performed treatments, the data from each experiment were analyzed by the using analysis of variance (ANOVA), followed by Least-Significant Difference (LSD) test (P = 0.05), using SAS ver. 9.1 statistical software. All the experiments were conducted in a completely randomized design in three replication. Graphs and figures were plotted using Excel program.

Data availability
All data that support the findings of this study are available from the corresponding author upon reasonable request.